The protozoan parasites of the genus Leishmania are responsible for causing leishmaniasis, a symptomatically complex disease which essentially affects men and animals in tropical and subtropical regions. It is estimated that the number of new cases of human visceral leishmaniasis can reach the number of 500,000, there being a minimum of several tens of millions of persons affected. Additionally the number of cutaneous and mucocutaneous leishmaniasis can be of the order of 2.000.000 per year (Modabber., 1990). Although the persons at risk of contracting the different types of leishmaniasis can be estimated in about 350 million, the number of persons with real infection can be much higher, due to the fact that there are no clear estimations of the real cases of asymptomatic infections and because of the existence of cryptic infections. In fact, leishmaniasis can be considered within the global context as an infection/disease of endemic nature, situated between the 4th and 5th place in the ranking of parasitic diseases with world-wide repercussion.
Three main forms of leishmaniasis can be distinguished: cutaneous, mucocutaneous and visceral, the characteristics of which mostly depend upon the localisation of the parasite, the species to which it belongs and the clinical manifestations it produces. The species distributed along Asia and certain regions in the Mediterranean area bring about the presence of the cutaneous form, with localised ulceration which, in many cases, heal spontaneously. These manifestations are caused by L. major and L. tropica. L. aethiopica (Mediterranean, Asia, Africa) also induces the cutaneous form of leishmaniasis, although its manifestation is more diffuse. In America, the species L. mexicana produces the cutaneous form with a generalised localisation that does not usually heal spontaneously. The mucocutaneous form of the disease in humans is caused by L. brasilieneis and is characterised by the presence of cutaneous lesions in oronasal and pharyngeal regions, bringing about the destruction of the mucosae. In America, Europe, Africa and Asia, the most frequent form of leishmaniasis is the visceral form, caused by L. chagasi, L. donovani and L. infantum. This form of leishmaniasis is characterised by clinical symptoms associated to fever, anaemia and an intense hepato/splenomegalia, which is lethal if it is not treated suitably at the right time. In the advanced form of the disease, the host is incapable of developing an effective immune response. All of these forms of leishmaniasis are also detected in canids and some rodents which, in fact, constitute the main reservoirs of the parasite. The health problem generated by L. infantum in the Mediterranean basin is serious because there is a high incidence of the infection/disease in dogs, and the vector insect is very extended. It is calculated that between 7% and 20% of all canids are infected by Leishmania, reaching 30%, in some areas of Spain where it is endemic. This fact constitutes a serious veterinary problem which additionally increases the risk of contagion, fundamentally by immunodepressed persons. In Europe, there are some 11 million dogs at risk of infection by Leishmania. 
L. infantum, like the rest of the species in the genus has a dimorphic biological cycle. The intermediate hosts are insects of the Psychodidae family, genus Phlebotomus. In the Mediterranean area of Europe, it has been demonstrated that the species P. arias and P. perniciosus are the main vectors, although the vectors P. papatosi, P. longicuspi and P. sergenti are also present. When the parasite is ingested by the vector together with the blood of a vertebrate host, it places itself in the gut in an extracellular form, it transforms into a promastigote and it divides. The infective forms migrate towards the pharynx and the proboscis, from where they will be inoculated into a new vertebrate host. The promastigotes are characterised in that they have a flagellum and an elongated shape of some 15 to 20 μm in length, with a rounded posterior end and a sharp anterior end. The nucleus is situated in central position and the kinetoplast at the anterior end. In culture media, the parasites exhibit a certain degree of morphological variability. After the inoculation of the promastigotes in the skin of the vertebrate host, the establishment, or not, of the infection depends essentially upon two factors: the existence of a suitable cell population—macrophages—and other cells of the phagocytic mononuclear system, and the ability of the parasite to survive and multiply itself in the interior of these cells.
The first step in the penetration of Leishmania into macrophages is the approximation and adherence to the plasma membrane of the target cell. In vitro studies seem to indicate that there is no direct chemotactic attraction of the promastigotes over the macrophages. Within the environment of tissues, free promastigotes activate complement by the alternative pathway, bringing about the formation of a concentration gradient of fraction C5a, which attracts macrophages and other inflammatory cells towards the site of inoculation. Once the promastigote is within a parasitophorous vacuole, the lysosomes fuse to it forming a phagolysosome. In infections caused by other micro-organisms, the phagolysosome is the organelle responsible for the lysis and elimination by means of several mechanisms such as the production of toxic oxygen radicals, by an oxidative metabolic process, the action of hydrolytic lysosomal enzymes, cationic proteins and low pH. The survival of the parasite in the phagolysosome is a function of its ability to resist and avoid said mechanisms.
The existence of an immune response against parasitisation by Leishmania which is both humoral and cellular was discovered from the first moments in which the disease was studied, and has been revised in numerous occasions. The type of humoral response depends upon the form of leishmaniasis. In the cutaneous form, the humoral response is very weak, whereas in the visceral type a high antibody response is observed. In the cutaneous affections there is a remarkable cell-mediated response, detectable both in vivo by means of delayed type hypersensitivity tests (DTH), as well as in vitro by means of lymphoblastic transformation tests and macrophage migration inhibition tests. In these cases the titre of serum antibodies is normally low and directly related to the seriousness of the process. Once the amastigotes are within the macrophages, the resolution of the infection depends essentially upon the cell-mediated immune mechanisms. The cellular response is determined by the joint action of macrophages, B cells, several sub-populations of T-cells, and the different lymphokines secreted by all of them. The parasitised macrophage processes the Leishmania antigens and expresses them on its surface by a process mediated by the class II Major Histocompatibility Complex (MHC-II), Additionally, the macrophage secretes IL-1, which acts as a second activating signal for the T-lymphocyte. In humans, visceral leishmaniasis or kala-azar is characterised by a weakened or absent cellular response, detectable both by the absence of delayed hypersensitivity (DTH) and by cell proliferation methods. Absence of proliferation of T-cells is detected even in the presence of mitogens such as concanavelin A or phytohaemagglutinin and inhibition in the production of IL-2 by stimulated T-cells.
In mice, the population of T-lymphocytes (CD4 phenotype) is heterogeneous and can be divided into at least two sub-populations according to the lymphokines they produce. These cells are essential in the development of protective immunity against cutaneous leishmaniasis (sub-population Th-1) and are at the same time involved in the suppression of the protective immune response (sub-population Th-2), T cells induced in resistant mice C57BL/6 or cured BALB/c mice are predominantly of the Th-1 type, whereas the cells in uncured BALB/c mice are of the Th-2 type. In general, the lymphokines secreted by these cells favour the development of the cell line which produces them and has an antagonic effect on the development of the other sub-population. Thus, IL-4 and IL-10 produced by Th-2 cells contribute to the progression of the infection, favouring the development of this line. Additionally, they can act directly on the macrophage, not permitting its activation. However, in mice susceptible of infection by Leishmania, deficient in the gene which encodes IL-4, contradictory results have been obtained. In some cases it has been observed that the absence of this cytokine redirects the response and increases the resistance to the infection whereas in others no differences are observed in the level of infection of the mice. In genetically resistant mice it has been observed that the absence of expression of the genes of IFN-γ or its receptor, CD40 or the ligand of CD40, increases the susceptibility to the infection. The interaction of CD40 and its ligand is necessary for the production of the cytokine TFN-γ necessary to direct the response towards Th-1. Mice with a resistant genetic base, which develop a Th-1 type response, become susceptible if they are deficient in the expression of IL-12, developing a Th-2 type response. Recent data suggests that the production of IL-12 is important to direct the response towards Th-1, and that the absence of IL-4 can avoid the Th-2 response.
There are a large number of Leishmania proteins which have an antigenic character essentially characterized by “Western Blot” methods. In the serum of patients and dogs infected by Leishmania it has been possible to detect the presence of antibodies against membrane proteins such as gp63, gp 46, PSA and KPM-11. Additionally several antigens of cytoplasmatic intracellular localisation have been characterised such as; Hsp70; Hsp83; LIP2a, LIP2b, LILIP0, H2A, H3 and a protein related to kinesin, K39 of L. chagasi. The reactivity of the antibodies, which recognise conserved proteins present in the serum of dogs infected by L. infantum is directed towards the least conserved areas of these proteins. Some of the membrane proteins are very antigenic in natural infections. In all the cases of natural infection there is a great restriction in the humoral response against the proteins because the antibodies developed during the infective process recognise very restricted areas of the same. The levels of IgG normally correspond to the intensity of the infection reflect the degree and the duration of antigenic stimulation determined by the parasitic load. A Leishmania antigen homologous to the type C kinase receptors (LACK) has recently been described which produces an early response of the Th-2 type. In mice transgenic for the LACK antigen and with a genetic background susceptible to the infection, the induction of tolerance to this antigen protects against the infection by Leishmania major. The anti-Leishmania antibodies can destroy the promastigotes in vitro in the presence of complement, promote phagocytosis and induce the adherence of several particles to the surface of promastigotes and amastigotes.
The pathologic reaction seems to go in parallel with the density of parasitised macrophages. In visceral leishmaniasis the macrophages generally distribute themselves in a diffuse manner throughout the different organic tissues. The type of inflammation is is constituted by an important cellular infiltrate with a predominance of lymphocytes and plamatic cells together with hyperplasia of phagocytic cells. The inflammation brings about alterations in the physiology of the affected organs producing serious systemic alterations. In some occasions local granulomatous inflammation occurs, with appearance of granulomae and microgranulomae in the different organs. These granulomae are constituted by macrophages and histiocytes (affected by parasites or not) surrounded by plasmatic cells and lymphocytes and, in some cases, by fibroblasts. This inflammatory process is accompanied by an organic reaction with the appearance of characteristic lesions in the affected organs. Some authors have found deposits of amyloid substance in virtually the totality of the organs. Some authors have formulated the hypothesis that the damages produced by the disease are not directly attributable to the aethiologic agent but to the organic reaction triggered.
Vaccine Against Leishmania 
The intense immunity which follows the recovery from cutaneous leishmaniasis has given a great impulse to the development of prophylactic vaccines against this disease. This immunity is derived from the induction of a T response which has associated to it the production of inflammatory cytokines which activate macrophages and destroy the parasites. The immunological memory in the cases of infection is probably maintained by the persistent presence of the parasite in the host in a process known as concomitant immunity.
The first studies regarding vaccination against Leishmania in the decade of the 40's used live parasites as immunogens. These studies led to the production of vaccines which produced significant protection against subsequent re-infection. However, the knowledge of the possibility that live organisms could produce real infections led to such vaccination programmes not to take place for very long and, on the contrary, interest focused upon vaccines based upon dead parasites. These studies provided the first evidence on the possibility of producing effective vaccines by inoculation of parasites.
The clinical trials which used immunisation with dead Leishmania promastigotes also began in the decade of the 40's. These vaccines yielded remarkable successes as a certain degree of protection was observed, which could oscillate between 0 and 82% depending on the population. These vaccines had a smaller effect than live parasite vaccines. The isolation of avirulent clones of L. major which protect mice against infection has also demonstrated that an attenuated vaccine is possible. However, the ignorance of the mutations which lead to the loss of virulence and the risk of production of virulent revertants make this type of vaccination currently unacceptable.
Recently there has been an important progress towards the identification of molecularly defined candidate vaccines, such as gp63, gp46/M2, the surface antigen related to the latter antigen known as PSA-2 and the proteins dp72 and gp70-2, the LACK protein and Kmp11. Specifically, the T epitopes present in protein gp63 have been identified, resulting in that only some of them are capable of inducing a T response, both of Th-1 as of Th-2. These antigens induce significant protection in model animals when they are administered with adjuvants. Protein PSA-2 of Leishmania is capable of protecting against infection by L, major by inducing a Th-1 response. To evaluate the mechanism of protection of this protein as a vaccine against Leishmania in humans, its ability to induce T-cell proliferation was studied, in patients which had suffered leishmaniasis and had recovered from it. It was observed that the protein is capable of bringing about a strong proliferation of the T-cells of these individuals, but not of controls with no prior history of infection. The response was of the Th-1 type, as was demonstrated by the cytokine induction pattern.
Sub-unit vaccines have focused strongly on protein antigens because they are easy to identify, isolate and clone. However, it is necessary to take into account that not all potential vaccine molecules have to be proteins. In fact, lipophosphoglycan (LPG) plays an essential role in the establishment of infection. Vaccination with LPG can protect against infection with L. major. In spite of the existence of a dogma which states that T-cells do not recognise non-proteinaceous antigens, the LPG molecule seems to be presented to T-cells by Langerhans cells of the skin. Additionally, there is evidence which has demonstrated that microbial glycolipids and other non-proteinaceous molecules can recognise T-cells when they are presented via the CD-1 route. Although there is no clear evidence that protein KMP11 associated to LPG induces a protective response, it has been proved that the proteinaceous fraction associated to LPG is capable of inducing a T response and IFN-γ, whereas the LPG fraction without protein is not.
It is normally accepted that sub-unit vaccines although protective, only induce a short term immunity. This problem may not be important in endemic areas, where the individuals can be periodically boosted by cause of natural infections. A major problem in the use of sub-units may arise from the fact that there may not be a response to a single antigen in a genetically diverse population. A cocktail of antigens containing B and T inducers may overcome this drawback. It has recently been published that an extract of membrane proteins of Leishmania infantum, when injected intraperitoneally, is capable of conferring protection against the virulent promastigote forms of this parasite, and that this protection is greater when the proteins are encapsulated in positively charged liposomes. Adjuvanticity and protective immunity were elicited by Leishmania donovani antigens encapsulated in positively charged liposomes.
Vaccination with nucleic acids carrying genes which encode Leishmania proteins involves the administration of genetic material of the parasite to the host. This DNA is taken up by the cells and is introduced into the nucleus where it is transcribed and subsequently translated in the cytoplasm. The advantage of this type of vaccination is that it is possible to direct the immune response by means of the MHC-I or the MHC-II route. The antigens produced intracellularly are processed in the cell and the peptides generated are presented on the cell surface in association with MHC-I molecules. The consequence would be that these molecules would give rise to the induction of cytotoxic T-cells. The antigens produced in an extracellular environment would be specially taken up by specialised antigen-presenting cells, processed and presented on their surface bound to MHC-II molecules, resulting in the induction and activation of CD4+ cells which secrete cytokines which regulate the effector mechanisms of other cells of the immune system.
The first DNA vector to be administered as a vaccine contained the gp63 gene. Also, the PSA-2 gene has been introduced into a plasmid and it has been observed that it generates a Th-1 response and induction of protection. Vaccination with DNA plasmids which contain Ag-2 induce a Th-1 response and protect against infection with L. major, while Ag-2 in stimulators immune complexes elicits a combined Th-1 and Th-2 response and does not protect despite the fact that IFN-γ is induced. Equally, the gene encoding the LACK protein has been administered subcutaneously to BALB/c mice, in an expression vector which expresses the protein under the control of the cytomegalovirus promoter, and protection against infection with L. major has been observed. In almost all cases in which DNA has been administered, the route has been intramuscular, although intradermal injection of particulate DNA must also be explored, as it requires a smaller amount of DNA. Other immunisation systems use vectors such as Salmonella, BCG or Vaccinia virus. It is interesting to remark that the inclusion of the gp63 gene in BCG is capable of inducing protection against L. major. 
The gene which encodes protein gp63 has also been introduced into gene delta araC under the control of the rac promotor in an attenuated Salmonella typhimurium. Oral administration of 1×109 colonies of the transformed bacterium induces a T response within the scope of both Th-1 and of cytotoxic cells against mastocytoma cells which express gp63. Protein gp63 in the form of gp63-ISCOMs complex induces protection in mice, evidenced by the reduction in inflammation and suppression of lesions. In serum, there are antibodies of the IgG2a type and, additionally, it is possible to observe a Th-1 response by induction of IL-2, IFN-γ and IL-10. No DTH response was observed. Salmonella typhi Nramp 1 transformed with gp63 elicits a Th-1 response with induction of IL-2 and IFN-γ, and a strong resolution of the lesions is detected. A protein of L. pifanoi known as P-4 induces significant protection against infection by Leishmania. Recent studies in humans with cutaneous leishmaniasis indicate that this protein or the peptides derived from it are capable of making T cells proliferate. There is no induction of IL-4, whereas IFN-γ is induced. Aro-A and aro-D mutants of Salmonella typhi transformed with IL-2, IFN-γ and TNF-α administered orally may serve as therapeutic Systems against infection by L. major. It is interesting to observe that in these patients there is a greater induction of iNOS. The gp&3 gene has also been cloned in Aro-A and Aro-D mutants, and it has been observed that after oral administration, the protein encoded is capable of inducing significant protection against infection with L. major. This same protein is capable of inducing protection when it is administered fused to several promoters in specific varieties (GID105 and GID106) of Salmonella.
An artificial protein denominated Q has recently been described by our group, which is composed of several antigenic fragments from 4 proteins of Leishmania infantum (more specifically, Lip2a, Lip2b, P0 and H2A), which, after being used as antigen, has proven to have an important value for the diagnosis of canine leishmaniasis, with a 93% sensitivity and a 100% specificity when compared with sera of control animals which are not infected. Equally, our group has demonstrated that protein hsp70 of Leishmania infantum is an important target of the immune response in infections caused by infection with this parasite.
With the object of exploring the possibility that protein Q may be used to design protection systems against infection by Leishmania infantum, both on its own as in combination with Hsp70, three series of experiments were designed using the hamster as a model. An experiment was designed to check whether immunisation with Protein Q protected the animals against infection on the short term, another to check whether immunisation protected them on the long term and the third was to check this protective effect after immunisation with the two proteins together. It was thereafter observed both from the short term analysis as well as from the long term analysis, that protein Q was capable of eliciting an immune response which reduces the parasitic load both in the Liver and in the Spleen after infection by Leishmania infantum in most of the immunised animals, and that immunisation with the proteins Q+Hsp70 also induced a significant response against both proteins and lead to a significant reduction of the parasitic load in most of the immunized animals.